Investigation of the GnRH antagonist degarelix isomerization in biological matrices

Abstract One of the main objectives of peptide drug design is the improvement of peptide pharmacokinetics with maintaining biological activity, which can be achieved by the complex modifications of the primary structure of the peptides. However, these changes often lead to the formation of peculiar impurities in the peptide drugs and their metabolites, which require the development of advanced analytical methods to properly assess their content. Here, we investigated the degradation of the potent long‐acting GnRH antagonist degarelix in various biologic media by the tailor‐made HPLC method, which allows precise determination of 5‐Aph(Hyd)‐degarelix isomer, an impurity found in the degarelix active pharmaceutical ingredient (API) during its manufacturing. Unexpectedly, we discovered a rapid and irreversible conversion of degarelix API into the corresponding hydantoin isomer in serum, suggesting that this impurity can be also a potential drug metabolite in vivo. This finding underlines the importance of the development of more accurate and performing analytical techniques to correctly characterize the chemical composition of the manufactured drugs and their behavior under physiological conditions.

antagonists currently used in clinical practice for prostate cancer treatment are derivatives of the natural decapeptide, where seven out of the ten residues are substituted with non-proteinogenic amino acids (Figure 1).
These substitutions strongly influenced the physicochemical and biological properties of the peptides. Starting from abarelix, the first GnRH antagonist approved by FDA, which showed noticeable allergic effects and was withdrawn lately from the US market, modifications of the natural GnRH led to the design of potent and long-acting inhibitors, such as azaline B and acyline. [9][10][11][12] Further modifications of the azaline B molecule resulted in the development of third generation GnRH antagonist degarelix with an improved pharmacological profile ( Figure 2). [13][14][15][16] After its approval by FDA in 2008, degarelix became the most widely used GnRH antagonist in patients with advanced prostate cancer. 17 The advantages of degarelix are its high affinity to the GnRH receptor, increased hydrophilicity, and decreased propensity to form gels. As a result, degarelix has better bioavailability than the previ- moieties, which afford multiple hydrogen-bonding sites and permit self-association with the formation of "amyloid" type fibrils, which dissolve over a long period (amyloid t 1/2 = 15 days). 18,19 However, the insertion of these non-proteinogenic amino acids poses several challenges for the manufacturing of degarelix. The Aph(Hor) moiety is known to be rapidly hydrolyzed under basic conditions to the Ncarbamoyl aspartyl intermediate with further rearrangement to the five-membered hydantoin isomer ( Figure 3). 20,21 Since the current standard manufacturing routes for peptide assembly rely on the solid-phase peptide synthesis (SPPS) approach, which requires the use of a base (usually piperidine) to achieve the deprotection of the FMOC group, the abovementioned rearrangement has to be considered unavoidable and the related impurity 5-Aph(Hyd)-degarelix (where Hyd = hydantoin) can be constantly present in the final degarelix active pharmaceutical ingredient (API). 22 For this reason, the potential formation of this impurity under physiological conditions plays a crucial role in the understanding of its potential toxicity profile. Indeed, degarelix binding to human serum albumin (HSA) is shown to be very high (76.3%) and the presence of many basic residues (58 lysines and 27 histidines) in the protein sequence and the binding sites could favor the isomerization process. [23][24][25] The detection and identification of metabolites are generally performed using HPLC-MS or Ultra-Performance Liquid Chromatography (UPLC) methods and it becomes very simple when the metabolite has a different molecular weight. On the other hand, 5-Aph(Hyd)-degarelix impurity has the exact molecular weight of the parent drug and, unfortunately, its proper quantification is complicated because of the high similarity of its structural and physio-chemical properties to those of the parent peptide ( Figure 2). Thus, the development of a tailored analytical method became necessary to properly evaluate its content in the degarelix API.
Previously, to evaluate the hydantoin impurity content in degarelix as an active pharmaceutical ingredient we developed an improved analytical method, which allows an excellent separation of degarelix and 5-Aph(Hyd)-degarelix, 26 and we applied it for the study of the stability of the peptide in various biological environments.

| MATERIAL S AND ME THODS
Analytical grade reagents and solvents were purchased in Merck and used without further purification. Degarelix API and 5-Aph(Hyd)-degarelix were prepared in Xingyin Pharmaceutical.
Human liver microsomes (pool of 50 male and female donors) were provided by Prolytic GmbH. Human male AB plasma (USA origin, sterile filtered) was obtained from Merck.

| Mass spectrometry analysis
Mass spectra were acquired on API 4000 spectrometer operating in the positive mode.

| Linearity investigation of the Analytical method A
The calibration standards were prepared in the same media, which were used for the stability tests. The calibration curves were in the range of 20-2000 ng/mL for degarelix and 5-Aph(Hyd)-degarelix and showed acceptable linearity over the calibration range for all media. The linearity of the Analytical method A was tested for the mixture of 5-Aph(Hyd)-degarelix and degarelix (2 μg/mL) for the concentration of the 5-Aph(Hyd)-degarelix 0.01 μg/mL, 0.02 μg/ mL, and 0.1 μg/mL. The stability of degarelix in the conditions of the Analytical method A was tested by dissolving it in the mobile phase at the concentration of 2 μg/mL and incubating for 2 h at 25°C.

| Stability investigations in human liver microsomes
The human liver microsome suspensions (protein concentration 20 mg/mL) were thawed for approximately 2 min in a water bath at RT and further kept on ice. After shaking the solution for 5-10 s, the required amount of microsomes was removed and the remaining sample was immediately re-frozen at approximately −80°C.
The samples were incubated at 37°C for 0 min (reference sample), and at different time intervals with or without 125 μL of 0.5 mg/ mL NADPH. In the case of the absence of NADPH 125 μL of the solution of 2 mg/mL glucose-6-phosphate and 0.45 μg/mL glucose-6-phosphate dehydrogenase in 2% NaHCO 3 were added. Following the addition of 500 μL acetonitrile, the samples were centrifuged and 50 μL of the supernatant of each sample were diluted with 20 μL of 0.2% formic acid in water and used for HPLC analysis.

| Data evaluation
The ANALYST software was used to integrate all peaks automatically

| RE SULTS
Several experiments performed in our laboratory demonstrated that the proper separation of the 5-Aph(Hyd)-degarelix isomer from degarelix is impossible with standard chromatographic methods with acidic mobile phases even when the UPLC method is applied.
On the contrary, the application of a basic mobile phase allows an excellent separation of the two peaks, allowing the adequate measurement of the content of this impurity in the degarelix API ( Figure 4, Table 1). 26 The farin. Furthermore, the same trend was observed when albumin solution was added with amino acids or ions normally present in the human plasma (data not shown). 28 The rate of formation of the 5-Aph(Hyd)-degarelix in human liver microsomes was much slower and it barely depended on the presence of NADPH, confirming that the absence of the involvement of the cytochrome P450 system in its formation ( Figure 5D). However, the concentration of the degarelix in solution was markedly reduced after 48 h (about 33% in the presence of NADPH), which indicates a metabolic pathway different from the dihydroorotate-hydantoin isomerization.
To evaluate the reversibility of the dihydroorotate-hydantoin isomerism, we also investigated the stability of 5-Aph(Hyd)-degarelix in the same testing environments (Figure 7). In human plasma, a gradual decrease of the 5-Aph(Hyd)-degarelix concentration was observed, which can be due to the degradation of the peptide or its aggregation in these conditions. No reverse formation of degarelix was detected over the period studied, confirming the irreversibility of the isomerism. On the contrary, in other matrices, we did not see any change in the 5-Aph(Hyd)-degarelix content.

F I G U R E 4
Comparison of degarelix API (red) and degarelix API with the addition of 0.5% (w/w) of the 5-Aph(Hyd)degarelix (black) using basic eluent (Analytical method A, top) and acidic eluent (Analytical method B, bottom) (see Materials and Methods for details).

| DISCUSS ION
Degarelix has emerged as a promising GnRH antagonist for prostate cancer treatment and its pharmacologic properties and metabolic pathways have been extensively studied during the past decade.
The previous studies of degarelix metabolism in humans showed that the peptide is excreted unchanged via renal pathway, but it is sequentially degraded by the hepatobiliary system. 29 Among the metabolites after 72 h mainly C-terminally truncated nonapeptide FE200486 (1-9)-OH was detected in plasma in the amount up to 6.3%. 29 In vitro studies showed that degarelix was not a substrate for the human cytochrome P450 system and only very minor in vitro degradation was observed after incubation of the peptide in liver microsomes. 30 However, in fresh hepatocytes, it was rapidly degraded to the nonapeptide metabolite. Thus, the origin of the nonapeptide in plasma samples could be due to the enzymatic degradation by endopeptidases located in the hepatic tissue. 29 Metabolite pattern study allowed the detection of N-terminal tetra-and penta-peptides as main fragments formed during the passage of the hepatobiliary system. 31 In previous studies of degarelix metabolism, chromatographic

ACK N OWLED G M ENTS
The authors have nothing to report.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors report no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article.

E TH I C S S TATEM ENT
This article does not contain any studies with humans or animal participants. There are no human participants in this article and informed consent is not applicable.